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Query: EC:1.14.99.3 (heme oxygenase)
 4,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A truncated, soluble, and enzymatically active rat heme oxygenase lacking its membrane-associative, C-terminal segment was expressed in E. coli strain JM109. The roles of its four histidine residues were examined by determining the enzymatic activities of mutant enzymes in which each of these residues in turn was replaced by alanine. Mutation of histidine residue 25 to alanine resulted in marked decrease in activity for heme breakdown, indicating that this histidine residue has an important role in the heme oxygenase reaction.
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PMID:Importance of histidine residue 25 of rat heme oxygenase for its catalytic activity. 154 Jan 95

A cDNA from a chick liver library that encodes for haem oxygenase has been cloned and sequenced. Positive clones were identified with monospecific antibodies to the purified enzyme from chick liver and a cDNA of rat haem oxygenase-1. The length of the cDNA is 1258 bases. An open reading frame of 888 bases was identified by comparison of nucleotide and amino acid sequences with those previously identified for haem oxygenase of mammalian or avian origin. The protein corresponding to this fragment of DNA is composed of 296 amino acid residues and has a molecular mass of 33,509 Da, which is similar to that previously estimated for haem oxygenase purified from chick liver. Unequivocal identification of this clone as that complementary to haem oxygenase was provided by (a) comparison of amino acid compositions and partial sequences with those previously established for the purified enzyme, (b) comparison with nucleotide and amino acid sequences for haem oxygenase from rat and human sources and (c) expression in Escherichia coli with production of high levels of mRNA, protein and haem oxygenase activity after exposure of the transfected bacteria to isopropyl beta-D-thiogalactopyranoside (IPTG). Overall, the similarity of chick haem oxygenase to rat and human haem oxygenase (nucleotides 66% and amino acids 62%) is moderately high. The region between proline-129 and alanine-157 is identical in all three enzymes, including histidine-135, which is proposed to play a key role in binding the substrate haem at the active centre of the enzyme. Northern blots also show that treatment of chicks with CdCl2, a potent inducer of haem oxygenase, results in increases in 1.65-1.70 kb mRNA, which hybridizes selectively to the full-length cDNA or to a synthetic 24-base oligonucleotide with sequence identical to that of a portion of the haem oxygenase cDNA. These results suggest that Cd-dependent induction of haem oxygenase is due to increased transcription of the gene or stabilization of its message.
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PMID:Cloning, sequencing and expression of cDNA for chick liver haem oxygenase. Comparison of avian and mammalian cDNAs and deduced proteins. 199 64

A truncated, soluble rat heme oxygenase-1 lacking its C-terminal, membrane-anchoring segment, and its His25-->Ala and His132-->Ala mutants have been prepared by site-directed mutagenesis and expression in Escherichia coli. We found that wild-type enzyme can degrade heme to biliverdin, but its specific activity was about one-fifth that of the native, full-length enzyme, suggesting that the C-terminal segment is important for accepting electrons from NADPH cytochrome P450 reductase. His132-->Ala mutant had an enzyme activity comparable to that of the wild-type enzyme; hence, the highly conserved His132 is not essential for the display of the heme oxygenase activity. In contrast, His25-->Ala mutation completely abolished the enzyme's catalytic activity. A five-coordinate type ferrous NO EPR spectrum was observed for the heme-heme oxygenase H25A complex. Hence, we conclude that His25 is the proximal axial ligand of the heme iron and is essential for the heme degradation activity of the enzyme.
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PMID:Demonstration that histidine 25, but not 132, is the axial heme ligand in rat heme oxygenase-1. 787 92

Cleavage of heme b (Fe-protoporphyrin IX) at the alpha-meso carbon bridge is catalyzed by heme oxygenase isozymes, HO-1 and HO-2, to form biliverdin IX alpha. Currently, we have examined the requirement for the amino terminus and the hydrophobic carboxy terminus of rat HO-2 expressed in Escherichia coli for heme degradation activity and have assessed the importance of His 151 for this activity. His 151 is in the longest span of amino acids (24 residues) which are present, with only a single conservative substitution, in seven cloned heme oxygenases including the apparent single isozyme in chicken. We show His 151 is essential for cleavage of heme, as substitution of alanine for this residue by site-directed mutagenesis resulted in expression of an inactive protein with immunoreactivity toward antibody to rat HO-2. A cDNA construct in which nucleotides encoding the 33 N-terminal amino acid residues were deleted, when expressed, produced a protein of predicted size and immunoreactivity with antibody to HO-2 but also devoid of heme degrading activity. The presence of additional residues at this terminus, for the most part, accounts for the larger size of HO-2 compared to HO-1. Conversely, the hydrophobic region at the carboxy terminus did not appear to be essential for heme degradation. A construct in which the sequence encoding the primarily hydrophobic amino acids of the carboxy terminus was replaced by a sequence encoding predominantly hydrophilic residues expressed a protein which retained full capability to convert heme to biliverdin. Further, the construct with a hydrophilic carboxy terminus was not appreciably associated with bacterial membranes, suggesting that the carboxy terminus in the wild-type protein serves as a membrane anchor for this enzyme.
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PMID:Domains of rat heme oxygenase-2: the amino terminus and histidine 151 are required for heme oxidation. 848 44

His-25 and His-132 are the primary candidates for the proximal heme iron ligand in heme oxygenase isozyme-1 (HO-1). The unambiguous spectroscopic demonstration that His-25 is the proximal iron ligand leaves the role of His-132 uncertain. Absorption and resonance Raman spectroscopy are used here to establish that mutation of His-132 to an alanine, glycine, or serine does not alter the histidine-iron bond, but results in the loss of the water molecule coordinated to the distal side of the iron in the wild-type enzyme-substrate complex. The His-132 mutations also (a) destabilize the ferrous-O2 complex with respect to autoxidation, which should result in partial uncoupling of NADPH consumption from heme oxidation, and (b) decrease the affinity of the enzyme for heme. The catalytic activity of the protein is decreased but not suppressed by these mutations: the H132G and H132A mutants retain 40-50% and the H132S mutant 20% of the activity of the wild-type protein. His-132, however, is required for catalytic turnover of the protein with H2O2. These results place His-132 close to the iron on the distal side of the heme pocket and indicate that His-132 facilitates, but is not absolutely required for, the catalytic turnover of HO-1.
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PMID:Heme oxygenase (HO-1): His-132 stabilizes a distal water ligand and assists catalysis. 854 75

Heme oxygenase is a key enzyme in the oxygen-dependent heme catabolism pathway. In order to clarify the role of highly conserved His132 in heme oxygenase isoform-1, we have prepared 30 kDa truncated rat heme oxygenase mutants in which His132 has been replaced by Ala, Gly, and Ser. The expressed recombinant mutant proteins were isolated in inclusion bodies and were recovered from the lysis pellet by dissolution in urea followed by dialysis. The solubilized fraction obtained, however, was composed of a mixture of a functional enzyme and an inactive fraction. The inactive fraction was removed by Sephadex G-75 gel filtration column chromatography, as it eluted out of the column at the void volume. The gel filtration-purified heme oxygenase mutants have spectroscopic and enzymatic properties identical to those of wild type. The hemin complex of the H132A mutant exhibits a transition between a high-spin acid form and a low-spin alkaline form with a pKa value of 7.6 identical to that in the wild-type complex. These results demonstrate that His132 in heme oxygenase does not link to the coordinated water molecule and is not an essential residue for the enzyme activity. These results are in accordance with our previous preliminary results [Ito-Maki, M., Ishikawa, K., Mansfield Matera, K., Sato, M., Ikeda-Saito, M., & Yoshida, T. (1995) Arch. Biochem. Biophys. 317, 253-258] but contradict a recent report that His132 is the distal base linked to the coordinated water molecule and an important residue for enzyme catalysis [Wilks, A., Ortiz de Montellano, P. R., Sun, J., & Loehr, T. M. (1996) Biochemistry 35, 930-936]. Prolonged storage of the solubilized fraction from the inclusion bodies of the mutants, H132S in particular, results in an increase in the void volume fraction with a concomitant decrease of the 30 kDa fraction. We infer that His132 plays a structural role in stabilization of the heme oxygenase protein.
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PMID:Histidine-132 does not stabilize a distal water ligand and is not an important residue for the enzyme activity in heme oxygenase-1. 912 12

The heme oxygenase (HO) system degrades heme to biliverdin and CO and releases chelated iron. In the primary sequence of the constitutive form, HO-2, there are three potential heme binding sites: two heme regulatory motifs (HRMs) with the absolutely conserved Cys-Pro pair, and a conserved 24-residue heme catalytic pocket with a histidine residue, His151 in rat HO-2. The visible and pyridine hemochromogen spectra suggest that the Escherichia coli expressed purified HO-2 is a hemoprotein. The absorption spectrum, heme fluorescence quenching, and heme titration analysis of the wild-type protein versus those of purified double cysteine mutant (Cys264/Cys281 --> Ala/Ala) suggest a role of the HRMs in heme binding. While the His151 --> Ala mutation inactivates HO-2, Cys264 --> Ala and Cys281 --> Ala mutations individually or together (HO-2 mut) do not decrease HO activity. Also, Pro265 --> Ala or Pro282 --> Ala mutation does not alter HO-2 activity. Northern blot analysis of ptk cells indicates that HO-2 mRNA is not regulated by heme. The findings, together with other salient features of HO-2 and the ability of heme-protein complexes to generate oxygen radicals, are consistent with HO-2, like five other HRM-containing proteins, having a regulatory function in the cell.
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PMID:Heme oxygenase-2 is a hemoprotein and binds heme through heme regulatory motifs that are not involved in heme catalysis. 913 9

A truncated, soluble, and enzymatically active form of human heme oxygenase-2 (DeltaHHO2) was expressed in Escherichia coli. To identify the axial heme ligand of HO-2, His-45 to Ala (DeltaH45A) and His-152 to Ala (DeltaH152A) mutants have been prepared using this expression system. DeltaH45A could form a 1:1 complex with hemin but was completely devoid of the heme degradation activity. A 5-coordinate-type ferrous NO EPR spectrum was observed for the heme-DeltaH45A complex. The DeltaH152A mutant was expressed as an inclusion body and was recovered from the lysis pellet by dissolution in urea followed by dialysis. The solubilized fraction obtained, however, was composed of a mixture of a functional enzyme and an inactive fraction. The inactive fraction was removed by Sephadex G-75 column chromatography since it eluted out of the column at the void volume. The gel filtration-purified DeltaH152A exhibited spectroscopic and enzymatic properties identical to those of wild-type. We conclude, in contrast to the previous reports (McCoubrey and Maines (1993) Arch. Biochem. Biophys. 302, 402-408; McCoubrey, W. K., Jr., Huang, T. J., and Maines, M. (1997) J. Biol. Chem. 272, 12568-12574), that His-45, but not His-152, in heme oxygenase isoform-2 is the proximal ligand of the heme and is essential for the heme degradation activity of the enzyme. His-152 appears to play a structural role in stabilization of the heme oxygenase protein.
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PMID:Identification of histidine 45 as the axial heme iron ligand of heme oxygenase-2. 946 79

The human heme oxygenase-1 crystal structure suggests that Gly-139 and Gly-143 interact directly with iron-bound ligands. We have mutated Gly-139 to an alanine, leucine, phenylalanine, tryptophan, histidine, or aspartate, and Gly-143 to a leucine, lysine, histidine, or aspartate. All of these mutants bind heme, but absorption and resonance Raman spectroscopy indicate that the water coordinated to the iron atom is lost in several of the Gly-139 mutants, giving rise to mixtures of hexacoordinate and pentacoordinate ligation states. The active site perturbation is greatest when large amino acid side chains are introduced. Of the Gly-139 mutants investigated, only G139A catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, but most of them exhibit a new H(2)O(2)-dependent guaiacol peroxidation activity. The Gly-143 mutants, all of which have lost the water ligand, have no heme oxygenase or peroxidase activity. The results establish the importance of Gly-139 and Gly-143 in maintaining the appropriate environment for the heme oxygenase reaction and show that Gly-139 mutations disrupt this environment, probably by displacing the distal helix, converting heme oxygenase into a peroxidase. The principal role of the heme oxygenase active site may be to suppress the ferryl species formation responsible for peroxidase activity.
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PMID:Replacement of the distal glycine 139 transforms human heme oxygenase-1 into a peroxidase. 1094 63

Fanconi anemia (FA) is an autosomal recessive disorder manifested by chromosomal breakage, birth defects, and susceptibility to bone marrow failure and cancer. At least seven complementation groups have been identified, and the genes defective in four groups have been cloned. The most common subtype is complementation group A. Although the normal functions of the gene products defective in FA cells are not completely understood, a clue to the function of the FA group A gene product (FANCA) was provided by the detection of limited homology in the amino terminal region to a class of heme peroxidases. We evaluated this hypothesis by mutagenesis and functional complementation studies. We substituted alanine residues for the most conserved FANCA residues in the putative peroxidase domain and tested their effects on known biochemical and cellular functions of FANCA. While the substitution mutants were comparable to wild-type FANCA with regard to their stability, subcellular localization, and interaction with FANCG, only the Trp(183)-to-Ala substitution (W183A) abolished the ability of FANCA to complement the sensitivity of FA group A cells to mitomycin C. By contrast, TUNEL assays for apoptosis after exposure to H2O2 showed no differences between parental FA group A cells, cells complemented with wild-type FANCA, and cells complemented with the W183A of FANCA. Moreover, semiquantitative RT-PCR analysis for the expression of the peroxide-sensitive heme oxygenase gene showed appropriate induction after H2O2 exposure. Thus, W183A appears to be essential for the in vivo activity of FANCA in a manner independent of its interaction with FANCG. Moreover, neither wild-type FANCA nor the W183A mutation appears to alter the peroxide-induced apoptosisor peroxide-sensing ability of FA group A cells.
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PMID:Functional analysis of the putative peroxidase domain of FANCA, the Fanconi anemia complementation group A protein. 1116 29


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